PROJECT SUMMARY Telomeres provide a sensitive sentinel of DNA damage. Containing only ~1/3000th of the total DNA in a human cell, they shelter us from oncogenic progression, monitor the number of cell divisions and respond to insult from environmental agents including UV, toxic metals, and organic pollutants. This is accomplished via damage signaling pathways which respond to the presence of damage in the telomeric DNA. Little is known about how DNA damage triggers structural changes in the telomere to activate damage signaling pathways. In 1999 we demonstrated that mammalian telomeres are arranged into large loops (t-loops); subsequently confirmed in numerous systems. In addition to the t-loop, the telomere is rich in complex structures including the t-loop junction, the single strand (ss) overhang, and the newly discovered telomeric R-loops-- all bound by the shelterin proteins. In our past grant cycle, we made a paradigm-shifting finding linking t-loop formation to telomere transcription. Transcription generates t-loops at high efficiency in model templates including ones the size of human telomeres. Transcription also leaves behind highly stable telomeric R-loops. R-loops can lead to genomic instability, gross chromosomal rearrangements, and cancer. We observed that telomeric R-loops are unusually stable and bind p53 tightly. R-loops may be the single greatest source of DNA damage at telomeres. These discoveries provide a wealth of new tools and approaches toward understanding telomere structure and its function as a damage sensor. We can now produce telomeric DNA >10 kb in length and we will apply biophysical and biochemical tools to further understand the structure of large telomeric single stranded (ss) DNA and telomeric RNA (TERRA). Approaches will include EM analysis combined with single molecule magnetic tweezers studies. The nature of the highly stable t-loop junction which also contains RNA will be examined using biochemical probes. We are poised to ask whether Rad51/52 alone or together with hnRNPA1 will insert TERRA RNA in trans into telomeric DNA to form telomeric R-loops. Employing our new affinity method for purifying telomeric DNA we will isolate telomeric DNA containing R-loops generated in vivo and map the distribution and size of the R-loops. Our guiding hypothesis is that t-loops evolved in part to provide a means to self-prime telomere extension and this may have preceded the evolution of telomerase. To test this, we will determine whether t-loop junctions generated by transcription can initiate replicative telomere extension. If so, this would mimic the ALT cancer phenotype and provide a new vision for the role of t-loops. BLM, WRN, and RTEL1 helicases act at the telomere and RTEL1 resolves t-loops in vivo. Using our large t-loop substrates generated by transcription and ones containing R-loops, we will examine the action of these proteins together with GEN1, Mus81-EME-1 and other nucleases on these highly relevant biological substrates, heretofore not available. The application of combined quantitative EM analysis with biochemical assays will be applied throughout.